African Journal of Pharmacy and Pharmacology Vol. 6(4), pp. 248-252, 29 January, 2012 Available online http://www.academicjournals.org/ajpp DOI: 10.5897/AJPP11.701 ISSN 1996-0816 ©2012 Academic Journals

Full Length Research Paper

Effects of rosmarinic acid on male sex hormones (testosterone-FSH-LH) and testis tissue apoptosis after exposure to electromagnetic field (EMF) in rats

Arash Khaki1*, S. A. M. Imani2 and Farhad S. Golzar1

1Department of Veterinary Pathology, Tabriz Branch, Islamic Azad University, Tabriz, Iran. 2Young Researchers Club, Tabriz Branch, Islamic Azad University, Tabriz, Iran.

Accepted 31 November, 2011

Rosmarinic acid (RA) is a polyphenol derived from many common herbal plants of the Lamiaceae group: Rosemary, sage, Spanish sage, oregano, basil, marjoram, thyme, the mint group, lavender, perilla and lemon balm. It is currently being studied for its effects on Alzheimer's disease and some other diseases. Rosmarinic acid in its natural state as part of a herb has been used to strengthen the memory and to improve mood by dispersing melancholy. Male Wistar rats (n = 40) were allocated to four groups, a control group (n = 10) and three treatment groups (n = 30). The first treatment group received rosmarinic acid (5 mg/kg body weight), the second extract group received rosmarinic acid (5 mg/kg body weight) and electromagnetic field (EMF) exposure at 50 Hz for 40 consecutive days, whilst the forth group received only EMF exposure for 40 consecutive days. Animals were maintained under standard conditions. At the conclusion of the test period, rat testes tissues were removed from all group members and animals were maintained under standard conditions. Tissue preparation was performed and analyzed for apoptosis. There was a significant increase in apoptosis in EMF group and significant increase in testosterone serum level in RA group when compared with other groups (P < 0.05). EMF has negative effect on testis histology in rats. However, these side effects are less seen in the EMF group that received RA. Therefore, it is recommended that usage of rosmarinic acid in modern country has fewer industrial side effects on male fertility.

Key words: Apoptosis, electromagnetic field (EMF), male fertility, rosmarinic acid, testis.

INTRODUCTION

The antioxidant capacity of phenolic compounds, improves the health of auditory regulation organs. An flavonoids and foods rich in these compounds, has been electromagnetic field (EMF or EM field) is a physical field repeatedly demonstrated in various in vitro and in vivo produced by electrically charged objects. EMFs affect the systems (Alexandopoulou et al., 2006). Rosmarinic acid behavior of charged objects in the vicinity of the field. The (RA), a water-soluble polyphenolic component isolated EMF extends indefinitely through space and determines from Dansam-Eum (DSE), has been reported to have electromagnetic interaction (Schüz et al., 2009; Emre et anti-oxidative (Huang and Zhang, 1992; Firdose et al., al., 2011). EMF is one of the four fundamental forces of 2011), anti-inflammatory (Osakabe et al., 2004) and anti- nature; the others are gravitation, the weak interaction depressive activities (Takeda et al., 2002). Li et al. (2007) and the strong interaction. The EMF can be viewed as a reported that RA was found in the brain following combination of an electric field and a magnetic field. The intravenous administration of Salvia miltiorrhiza. Hence, increased use of power lines and modern electrical RA can be considered as an active compound that devices is of concern as a public health hazard, and chronic exposure to EMF has attracted considerable attention. Exposure to EMF adversely affects spermatogenesis by the Sertoli and Leydig cells *Correspondence author. E-mail: [email protected]. Tel: (Martínez-Sámano et al., 2010). Magnetic fields of 50 Hz +98-914313-8399. also induce cytotoxic and cytostatic changes in the Khaki et al. 249

differentiating spermatogonia of mice (Khaki et al., 2008). administered intra-peritoneally as an anesthetic, and the peritoneal Little is known about the effect of EMF on the cavity was opened with a lower transverse abdominal incision. The cytoarchitecture of the boundary tissue of the testes were then immediately removed from the control and experimental groups. The weight of the testes for each group seminiferous tubules, which perform a number of crucial member was recorded. Animals were then decapitated between functions, including, mechanical support and transport of 10:00 and 12:00 h. At the end of 4 weeks of treatment, testis was nutrients to the spermatozoa (Roychoudhury et al., 2009) dissected from each rat, 24 h after the last administration. Then and sperm discharge by maintaining pressure on the tissue preparation was performed to investigate, vein congestion tubules (Iorio et al., 2007). Other studies have and apoptosis by TUNEL. investigated transitory effects of EMF on the testes, but no previous study has reported the possibility of recovery TUNEL analysis of apoptosis from the potentially harmful effects of EMF exposure after an exposure-free period. Furthermore, studies by Khaki The in-situ DNA fragmentation was visualized by TUNEL method et al. (2011) showed that many antioxidants can (Khaki et al., 2008). Briefly, dewaxed tissue sections were moderate harmful effects of EMF waves in testis tissues. predigested with 20 mg/ml proteinase K for 20 min and incubated in phosphate buffered saline (PBS) solution containing 3% H2O2 for The present study was designed to investigate the 10 min to block the endogenous peroxidase activity. The sections protective effects of RA on EMF effects on testis were incubated with the TUNEL reaction mixture, fluorescein-dUTP apoptosis. (in situ Cell Death Detection, POD kit, Roche, Germany) for 60 min at 37°C. The slides were then rinsed three times with PBS and were incubated with secondary anti-fluorescein-POD-conjugate for 30 min. After washing three times in PBS, diaminobenzidine-H2O2 MATERIALS AND METHODS (DAB, Roche, Germany) chromogenic reaction was added on sections and counterstained with hematoxylin. As a control for A total of 40 male Wistar rats were maintained for use in this study. method specificity, the step using the TUNEL reaction mixture was Rats were housed together (10 per cage) and fed on a compact diet omitted in negative control serial sections, and nucleotide mixture in in the form of granules and water. The diet contained all the reaction buffer was used instead. Apoptotic germ cells were essential ingredients, including, vitamins and minerals. The quantified by counting the number of TUNEL stained nuclei per environmental conditions (temperature and humidity) in all the seminiferous tubular cross section. Cross sections of 100 tubules animal holding areas were continuously monitored. Temperature per specimen were assessed and the mean number of TUNEL was maintained in the range of 23°C and humidity was maintained positive germ cells per tubule cross-section was calculated. at 35 to 60%. Light was provided on a 12 h light/dark cycle from 0700 to 1900 h. All animals were treated in accordance to the Principles of Laboratory Animal Care (NIH). The experimental protocol was approved by the Animal Ethics Committee in Statistical analysis accordance with the guide for the care and use of laboratory animals prepared by Tabriz University of Medical Sciences. Rats Data were expressed as means with standard error of mean (SEM). were allocated to four groups, a control group (n = 10) and three Analyses of detected parameters were done by one-way ANOVA treatment groups (n = 30). The first treatment group received test followed by Tukey’s (honestly significant difference (HSD) test. rosmarinic acid (5 mg/kg body weight), the second extract group P-value < 0.05 was considered to be significant. received rosmarinic acid (5 mg/kg body weight) and EMF exposure at 50 Hz for 40 consecutive days, while the forth group received only EMF exposure for 40 consecutive days. Animals were RESULTS maintained under standard conditions.

Compared to the control group, apoptotic cells EMF-producing system percentage decreased following administration of Ocimum basilicum extract (1.5 g/kg body weight). The equipment was based on the Helmholtz coil, which operated Exposure to 50 Hz of EMF caused a significant increase following Fleming’s right hand rule. The equipment produced an in the apoptotic cells percentage. When 50 Hz of EMF alternating current of 50 Hz, which created an EMF of 80 G. The was administrated together with O. basilicum extract (1.5 intensity of the EMF was controlled using a transformer. The equipment had two main parts. In the first part, there were two g/kg body weight), apoptotic cells percentage decreased copper coils that were placed, one above the other and separated significantly (P < 0.05) from 18.12 ± 1.05 to 10.05 ± 0.01 by a distance of 50 cm. A cylindrical wooden vessel was placed in spermatogonia, and decreased significantly (P < 0.05) between the coils (the exposure area), the interior of which from 20.11 ± 0.05 to 9.05 ± 0.05 in primary contained a chamber for holding the caged experimental animals. spermatocytes, respectively, vein congestion decreased The second part was the transformer, which controlled the input and output voltage using a voltmeter and the current with an significantly (P < 0.05) from 8 ± 0.03 to 0.5 ± 0.01. These ampere meter. A fan was used as required, to prevent increase in results indicate the protective effect of O. basilicum temperature inside the chamber. Four cages at a time were placed against EMF-induced apoptosis (Table 1). within the chamber, with ten rats per cage.

Surgical procedure DISCUSSION

On day 40, a sodium pentobarbitall solution (40 mg/kg) was Plants and natural products are extensively used in 250 Afr. J. Pharm. Pharmacol.

Table 1. Means of germinal cells apoptosis and male sex hormones (Testosterone-FSH-LH) of rats testis exposed to EMF and rosmarinic acid.

EMF Rosmarinic acid Rosmarinic acid + Groups Control (50 Hz) (5 mg/kg body weight) EMF Spermatogonia apoptotic cell (%) 8 ± 0.05 22.11 ± 0.05* 4.25 ± 0.03 12.05 ± 0.02* Primary spermatocytes apoptotic cell (%) 5 ± 0.05 19.02 ± 0.05* 3.75 ± 0.01 10.05 ± 0.05* Testis weight’s(Gram) 1.40 ± 0.821 1.10 ± 0.001 1.49 ± 0.001 1.39 ± 0.371 Testis vein congestion (%) 1 ± 0.01 10 ± 0.02* 6 ± 0.03 0.5 ± 0.01* Serum testosterone levels (ng/ml) 1.70 ± 0.01 1 ± 0.01 2.99 ± 0.210** 1.70 ± 0.01 LH levels (ng/ml) 1.51 ± 0.138 0.91 ± 0.128 1.73 ± 0.164 1.31 ± 0.128 FSH levels (ng/ml) 20.17 ± 1.005 18.17 ± 1.005 20.17 ± 1.005 21.17 ± 1.005

Data are presented as mean ± SE. *Significantly different at P < 0.05 level (compared with the control group). **Significantly different at P < 0.001 level (compared with the control group).

several traditional systems of medicine, so, screening The ability of antioxidants, such as ascorbic acid in these products for radio-protective compounds has semen to protect spermatozoa from oxidative damage several advantages, because they are usually considered has been shown by some authors (Timmermans, 1989). non-toxic and are widely accepted by humans. Many The main pharmacological actions of ginger and natural antioxidants, whether consumed before or after compounds isolated include immuno-modulatory, anti- radiation exposure, can confer some level of tumorigenic, anti-inflammatory, anti-apoptotic, anti- radioprotection. In addition to beneficial effects accrued hyperglycemic, anti-lipidemic and anti-emetic action. from established antioxidants, such as, vitamin C and E, Ginger is a strong antioxidant substance and may either and their derivatives, vitamin A, beta carotene, curcumin, mitigate or prevent generation of free radicals. It is Allium cepa, quercetin, caffeine, chlorogenic acid, ellagic considered a safe herbal medicine with only few and acid and bixin, protection is also conferred by several insignificant adverse/side effects (Dawson et al., 1992). novel molecules, including, flavonoids, epigallocatechin, Oxidants and antioxidants have attracted widespread and other polyphenols (Kirtikar and Basu, 1991; Khaki et interest in nutrition research, biology and medicine. It has al., 2009a, b, c). Fertility is one of the major problems in a become clear that constant generation of pro-oxidants, man’s life; about 25 and 35% of infertility is regarded to including oxygen free radical, is an essential attribute of be man and woman receptivity (Mosher and Pratt 1991). aerobic life (Acharya et al., 2008). A disturbance in the The importance of many of these factors is not yet clearly pro-oxidant/antioxidant system has been defined as understood. A better understanding of underlying oxidative stress. Reactive oxygen species (ROS) are very mechanisms in fertility and better study results clarifying reactive molecules ranked as free radicals owing to the the effectiveness of nutritional and biochemical factors presence of one unpaired electron, such as a superoxide are important to improve diagnosis and treatment. Smart ion, nitrogen oxide and hydroxyl radical, administration of choices for better foods might prevent body from many this extract with gentamicin was also able to diseases (Reddy et al., 2006; Suryavathi et al., 2005). As counterbalance the negative effect of gentamicin on all spermatogenesis stages occur in seminiferous tubule sperm count. However, oxidative stress occurs from an of testis, it is possible to evaluate the extent of imbalance between ROS and antioxidant actions. During spermatogenesis by the determination of the number of chronic oxidative stress caused by environmental factors spermatozoa produced per one gram of testicular (that is, UV light, ionizing radiation and toxic substances), parenchyma (Acharya et al., 2008; Hew et al., 1993). The infections, diabetes or lack of dietary antioxidants, an sperm count is considered as important parameter to inequity of cellular reducing equivalents capable of assess the effects of chemical on spermatogenesis detoxifying the increased burden of ROS has marked (Yousef, 2005). It has also been reported that there is a effects on normal cellular processes. However, in times direct correlation between the epididymal sperm count of oxidative stress, normal cellular respiration is also, still and motility with fertility in animals (Dawson et al., 1992; functioning, resulting in dysregulated mitochondrial free Timmermans, 1989; Yu et al., 2005). The oxidative radical production and disparity between ROS generation damage, elevated lipid peroxidation and the alteration of and antioxidant defenses (Schriner et al., 2005; Limón et membrane properties can lead to germ cell death at al., 2009). A previous study showed that 2 h of 60 Hz different stages of development and the sperm count EMF exposure immediately altered the metabolism of decrease (Bestas et al., 2006). Accordingly, it is expected free radicals, decreased superoxide dismutase (SOD) that antioxidant therapy acts as a protective defense activity in plasma, decreased glutathione (GSH) content against oxidative stress and improve fertility parameters. in the heart and kidney, but did not induce immediate lipid Khaki et al. 251

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The results causes disruption of tight junction-associated microfilaments in rat of other study showed the ability of O. basilicum in the Sertoli cells. Biol. Reprod., 49: 9-840. enhancement of protective effects of rats resulting from Huang YS, Zhang ZT (1992). Antioxidative effect of three water-soluble components isolated from Salviae miltiorrhizae in vitro. Acta. Pharm. decrease of apoptosis in testis, testis weights increasing, Sin., 27: 96–100. vein congestions decreasing and also increased Iorio R, Scrimaglio R, Rantucci E, Delle Monache S, Di Gaetano A, testosterone levels (Khaki et al., 2011). Our previous Finetti N, Francavilla F, Santucci R, Tettamanti E, Colonna R (2007). study, demonstrated that the administration of O. A preliminary study of oscillating electromagnetic field effects on human spermatozoon motility. 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